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QuantiNova Reverse Transcription Kit

For fast cDNA synthesis and reproducible real-time two-step RT-PCR

 

  • Eliminate artifacts using the gDNA Removal Mix
  • Monitor successful cDNA synthesis using the internal control
  • Synthesize cDNA over a wide linear range with high-affinity transcriptase
The unique QuantiNova Reverse Transcription Kit provides a fast and convenient procedure for cDNA synthesis with integrated genomic DNA removal and an additional internal control. Genomic DNA contamination in RNA samples is effectively eliminated by the gDNA Removal Mix, and the internal control RNA can be used to test successful reverse transcription and amplification. The QuantiNova Reverse Transcription Kit provides everything you need for fast and efficient reverse transcription. The synthesized cDNA is optimized for use in real-time PCR, allowing reliable quantification of targets from all regions of mRNA transcripts.
Cat No./ID: 205410
QuantiNova Reverse Transcription Kit (10)
$121.00
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For 10 x 20 µl reactions: 20 µl 8x gDNA Removal Mix, 10 µl Reverse Transcription Enzyme, 40 µl Reverse Transcription Mix (containing RT primers), 20 µl Internal Control RNA, 1.9 ml RNase-Free Water
Cat No./ID: 205411
QuantiNova Reverse Transcription Kit (50)
$430.00
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For 50 x 20 µl reactions: 100 µl 8x gDNA Removal Mix, 50 µl Reverse Transcription Enzyme, 200 µl Reverse Transcription Mix (containing RT primers), 100 µl Internal Control RNA, 1.9 ml RNase-Free Water
Cat No./ID: 205413
QuantiNova Reverse Transcription Kit (200)
$1,438.00
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For 200 x 20 µl reactions: 4 x 100 µl 8x gDNA Removal Mix, 4 x 50 µl Reverse Transcription Enzyme, 4 x 200 µl Reverse Transcription Mix (containing RT primers), 4 x 100 µl Internal Control RNA, 4 x 1.9 ml RNase-Free Water
Cat No./ID: 205813
QuantiNova IC Probe Assay (200)
$286.00
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400 µl primer / probe mix (10x) for 200 reactions, detects Internal Control RNA; use with QuantiNova Probe PCR Kit or QuantiNova Probe RT-PCR Kit
Cat No./ID: QT02589307
QuantiNova IC SYBR Green Assay
QuantiTect Primer Assay for SYBR-based detection of QuantiNova Internal Control RNA; available via GeneGlobe (sufficient for approx. 500 x 20 µl rxn). For use with QuantiNova SYBR Green PCR Kit or QuantiNova SYBR Green RT-PCR Kit
The QuantiNova Reverse Transcription Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Efficient removal of contaminating gDNA ensures precise quantification of transcripts.
Real-time two-step RT-PCR analysis of ACTB, with and without gDNA removal step. Instead of using RNA, genomic DNA was purified from human whole blood and used as template. Different amounts of gDNA (100 ng, 10 ng, 1 ng) were spiked into the cDNA synthesis reactions using the QuantiNova Reverse Transcription Kit. Real-time PCR was performed in triplicate on an Applied Biosystems 7500 Fast Real-Time PCR System using a 1:10 cDNA dilution and the QuantiNova Probe PCR Kit. Without gDNA removal, genomic DNA contamination resulted in a strong signal. This signal was successfully eliminated by the integrated gDNA removal step (N.D. = not detected).
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Internal control reliably detects presence of inhibitors.
Real-time two-step RT-PCR analysis of various target genes and the QuantiNova Internal Control RNA (QN IC RNA). One microgram of total RNA was purified from human whole blood, and the QN IC RNA was added to the RT reaction. RNA samples with inhibitor (0.003% sodium dodecyl sulfate) and without were tested in parallel. Real-time PCR was performed in triplicate on a Bio-Rad CFX384 using a 1:100 cDNA dilution and the QuantiNova SYBR Green PCR Kit. Resulting Cq shifts for the internal control and endogenous target transcripts were comparable, demonstrating that the IC RNA can be used to detect the presence of inhibitors.
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Precise quantification from 5 µg – 10 pg RNA.
Real-time two-step RT-PCR analysis of RPS27A. Total RNA was purified from human whole blood. cDNA was then synthesized from a serial dilution of RNA (5 µg – 10 pg) using the QuantiNova Reverse Transcription Kit. Real-time PCR was performed in triplicate on a Bio-Rad CFX384 using a 1:100 cDNA dilution and the QuantiNova Probe PCR Kit. The amplification plot shows a high dynamic range of the cDNA synthesis for the whole tested range (5 µg – 10 pg) of total RNA.
Performance
Contaminating genomic DNA in RNA samples is effectively and rapidly removed with the unique gDNA Removal Mix. Elimination of genomic DNA is crucial for accurate gene expression results and when design of RNA-specific primers or probes is not always possible, for example, when analyzing single-exon genes. With gDNA Removal Buffer, time is saved and costs are reduced, since a separate DNase digestion during or after purification of RNA samples is not required.

The newly developed internal control is a defined transcript (RNA molecule) that can simply be added to your samples and transcribed into cDNA. It is intended to report instrument or chemistry failures, errors in assay setup and the presence of inhibitors. The expected range of Cq (CT) values can be used to test successful reverse transcription/amplification and the reproducibility of your results.

The high RNA affinity of the QuantiNova Reverse Transcriptase enables high yields of cDNA from any RNA template. Difficult templates, such as those with high GC-content or complex secondary structure, are also successfully reverse transcribed. The Reverse Transcription Mix contains a specially optimized mix of oligo-dT and random primers that enable cDNA synthesis from all regions of RNA transcripts, including 5' regions. The kit provides high yields of cDNA template for real-time PCR analysis, regardless of where the amplified target region is located on the transcript, and provides greater sensitivity in the detection of low-abundance genes.

The QuantiNova Reverse Transcription Kit offers accurate results over a wide range of input amounts, including up to 5 µg RNA in the standard protocol, which can be doubled to accommodate exceptionally large amounts of RNA.
Principle
The QuantiNova Reverse Transcriptase has a high affinity for RNA and is capable of cDNA synthesis from a wide range of RNA amounts (10 pg – 5 μg) (see figure “Precise quantification from 5 µg – 10 pg RNA”). The kit provides high yields of cDNA template for real-time PCR analysis, regardless of where the amplified target region is located on the transcript. Even difficult templates, such as those with high GC-content or complex secondary structure, are successfully reverse transcribed. The QuantiNova RT Buffer has also been optimized to be compatible with real-time PCR buffers.

To obtain accurate results in real-time RT-PCR gene expression assays, it is important that only cDNA is amplified and detected. Interference by genomic DNA can effectively and rapidly be removed using the gDNA Removal Mix (see figure “Efficient removal of contaminating gDNA ensures precise quantification of transcripts”). Time is saved and costs are reduced, since a separate DNase digestion during or after purification of RNA samples is not required. Also, designing RNA-specific primers or probes is not necessary.

Detecting variations in cDNA synthesis allows you to check the reproducibility of your results. The newly developed internal control is a defined transcript (RNA molecule) that can be optionally added to your samples and transcribed into cDNA. It is intended to report instrument or chemistry failures, errors in assay setup and the presence of inhibitors. Because the internal control behaves comparable to real transcripts, it can be used to confirm successful reverse transcription and amplification in subsequent qPCR (see figure “Internal control reliably detects presence of inhibitors”).
 
Components of the QuantiNova Reverse Transcription Kit
Component Benefits
gDNA Removal Mix Detection of RNA only in real-time RT-PCR
Internal Control RNA Verification of successful RT-PCR performance
QuantiNova Reverse Transcriptase Use of a wide range of RNA amounts (10 pg – 5 μg RNA)
High sensitivity
QuantiNova RT buffer system Read-through of difficult templates
RT Primer Mix cDNA synthesis from all regions of transcripts, even from 5' regions
Procedure
Genomic DNA removal and cDNA synthesis take only 20 minutes with the QuantiNova Reverse Transcription Kit. The kit includes everything you need for fast cDNA synthesis.

Purified RNA is briefly incubated in gDNA Removal Mix to effectively remove contaminating genomic DNA. In contrast to other methods, the RNA sample is then used directly for reverse transcription in combination with a master mix prepared from the Reverse Transcription Mix plus Reverse Transcription Enzyme. With the QuantiNova Reverse Transcriptase, RNA can be transcribed at low temperatures, even through complex 2° degree structure. This ensures that the RNA will stay intact as the reaction takes place at 42°C and is then inactivated at 85°C. Additional steps for RNA denaturation or RNase H digestion are not necessary.
Applications
The QuantiNova Reverse Transcription Kit allows highly efficient and sensitive real-time RT-PCR for all types of starting materials, including laser-microdissected samples and tissue biopsies.
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